Disturbance in the protein landscape of cochlear perilymph in an Alzheimer's disease mouse model.

Hearing loss is a pivotal risk factor for dementia. It has recently emerged that a disruption in the intercommunication between the cochlea and brain is a key process in the initiation and progression of this disease. However, whether the cochlear properties can be influenced by pathological signals associated with dementia remains unclear. In this study, using a mouse model of Alzheimer's disease (AD), we investigated the impacts of the AD-like amyloid ß (Aß) pathology in the brain on the cochlea. Despite little detectable change in the age-related shift of the hearing threshold, we observed quantitative and qualitative alterations in the protein profile in perilymph, an extracellular fluid that fills the path of sound waves in the cochlea. Our findings highlight the potential contribution of Aß pathology in the brain to the disturbance of cochlear homeostasis.

Molecular bases of rod and cone differences.

In the vertebrate retina, rods and cones both detect light, but they differ in functional aspects such as light sensitivity and temporal resolution, and in some cell biological aspects. For functional aspects, both types of photoreceptors use a phototransduction cascade, consisting of a series of enzymatic reactions, to convert photon capture to an electrical signal. To understand the mechanisms underlying the functional differences between rods and cones at the molecular level, we compared biochemically, each of the reactions in the phototransduction cascades of rods and cones using the cells isolated and purified from carp retina. Although the cascade proteins are identical or are functionally similar between rods and cones, their activities together with their expression levels are mostly different. In general, reactions that generate a response are somewhat less effective in cones than in rods, but each of the reactions for termination and recovery of a response are much more effective in cones. These findings explain lower light sensitivity and briefer light responses in cones than in rods. In addition, our considerations suggest that a Ca2+-binding protein, S-modulin or recoverin, has a currently unnoticed role in shaping light responses. Upon comparison of the expression levels of proteins and/or mRNAs using purified cells, several proteins were found to be specifically or predominantly expressed in cones. These proteins will be of interest in future studies aimed at characterizing the differences between rods and cones.

Transducin activates cGMP phosphodiesterase by trapping inhibitory γ subunit freed reversibly from the catalytic subunit in solution.

Activation of cGMP phosphodiesterase (PDE) by activated transducin α subunit (Tα*) is a necessary step to generate a light response in vertebrate photoreceptors. PDE in rods is a heterotetramer composed of two catalytic subunits, PDEα and PDEß, and two inhibitory PDEγ subunits, each binding to PDEα or PDEß. Activation of PDE is achieved by relief of the inhibitory constraint of PDEγ on the catalytic subunit. In this activation mechanism, it is widely believed that Tα* binds to PDEγ still bound to the catalytic subunit, and removes or displaces PDEγ from the catalytic subunit. However, recent structural analysis showed that the binding of Tα* to PDEγ still bound to PDEα or PDEß seems to be difficult because the binding site of PDEγ to PDEα or PDEß overlaps with the binding site to Tα*. In the present study, we propose a novel activation mechanism of PDE, the trapping mechanism, in which Tα* activates PDE by trapping PDEγ released reversibly and spontaneously from the catalytic subunit. This mechanism well explains PDE activation by Tα* in solution. Our further analysis with this mechanism suggests that more effective PDE activation in disk membranes is highly dependent on the membrane environment.

Purification of cone outer segment for proteomic analysis on its membrane proteins in carp retina.

Rods and cones are both photoreceptors in the retina, but they are different in many aspects including the light response characteristics and, for example, cell morphology and metabolism. These differences would be caused by differences in proteins expressed in rods and cones. To understand the molecular bases of these differences between rods and cones, one of the ways is to compare proteins expressed in rods and cones, and to find those expressed specifically or dominantly. In the present study, we are interested in proteins in the outer segment (OS), the site responsible for generation of rod- or cone-characteristic light responses and also the site showing different morphology between rods and cones. For this, we established a method to purify the OS and the inner segment (IS) of rods and also of cones from purified carp rods and cones, respectively, using sucrose density gradient. In particular, we were interested in proteins tightly bound to the membranes of cone OS. To identify these proteins, we analyzed proteins in some selected regions of an SDS-gel of washed membranes of the OS and the IS obtained from both rods and cones, with Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) using a protein database constructed from carp retina. By comparing the lists of the proteins found in the OS and the IS of both rods and cones, we found some proteins present in cone OS membranes specifically or dominantly, in addition to the proteins already known to be present specifically in cone OS.

Effects of NDRG1 family proteins on photoreceptor outer segment morphology in zebrafish.

Rods and cones are functionally and morphologically distinct. We previously identified N-myc downstream-regulated gene 1b (ndrg1b) in carp as a cone-specific gene. Here, we show that NDRG1b and its paralog, NDRG1a-1, contribute to photoreceptor outer segment (OS) formation in zebrafish. In adult zebrafish photoreceptors, NDRG1a-1 was localized in the entire cone plasma membranes, and also in rod plasma membranes except its OS. NDRG1b was expressed specifically in cones in the entire plasma membranes. In a developing retina, NDRG1a-1 was expressed in the photoreceptor layer, and NDRG1b in the photoreceptor layer plus inner nuclear layer. Based on our primary knockdown study suggesting that both proteins are involved in normal rod and cone OS development, NDRG1a-1 was overexpressed or NDRG1b was ectopically expressed in rods. These forced-expression studies in the transgenic fish confirmed the effect of these proteins on the OS morphology: rod OS morphology changed from cylindrical to tapered shape. These taper-shaped rod OSs were not stained with N,N'-didansyl cystine that effectively labels infolded membrane structure of cone OS. The result shows that rod OS membrane structure is preserved in these taper-shaped OSs and therefore, suggests that tapered OS morphology is not related to the infolded membrane structure in cone OS.

ES1 is a mitochondrial enlarging factor contributing to form mega-mitochondria in zebrafish cones.

Total mass of mitochondria increases during cell proliferation and differentiation through mitochondrial biogenesis, which includes mitochondrial proliferation and growth. During the mitochondrial growth, individual mitochondria have been considered to be enlarged independently of mitochondrial fusion. However, molecular basis for this enlarging process has been poorly understood. Cone photoreceptor cells in the retina possess large mitochondria, so-called mega-mitochondria that have been considered to arise via the enlarging process. Here we show that ES1 is a novel mitochondria-enlarging factor contributing to form mega-mitochondria in cones. ES1 is specifically expressed in cones and localized to mitochondria including mega-mitochondria. Knockdown of ES1 markedly reduced the mitochondrial size in cones. In contrast, ectopic expression of ES1 in rods significantly increased both the size of individual mitochondria and the total mass of the mitochondrial cluster without changing the number of them. RNA-seq analysis showed that ERRα and its downstream mitochondrial genes were significantly up-regulated in the ES1-expressing rods, suggesting facilitation of mitochondrial enlargement via ERRα-dependent processes. Furthermore, higher energy state was detected in the ES1-expressing rods, indicating that the enlarged mitochondria by ES1 are capable of producing high energy. ES1 is the mitochondrial protein that is first found to promote enlargement of individual mitochondria.

Dephosphorylation during bleach and regeneration of visual pigment in carp rod and cone membranes.

On absorption of light by vertebrate visual pigment, the chromophore, 11-cis retinal, is isomerized to all-trans retinal to activate the phototransduction cascade, which leads to a hyperpolarizing light response. Activated pigment is inactivated by phosphorylation on the protein moiety, opsin. Isomerized all-trans retinal is ultimately released from opsin, and the pigment is regenerated by binding to 11-cis retinal. In this pigment regeneration cycle, the phosphates incorporated should be removed in order that the pigment regains the capability of activating the phototransduction cascade. However, it is not clear yet how pigment dephosphorylation takes place in the regeneration cycle. First in this study, we tried to estimate the dephosphorylation activity in living carp rods and cones and found that the activity, which is present mainly in the cytoplasm in both rods and cones, is three times higher in cones than in rods. Second, we examined at which stage the dephosphorylation takes place; before or after the release of all-trans retinal, during pigment regeneration, or after pigment regeneration. For this purpose we prepared three types of phosphorylated substrates in purified carp rod and cone membranes: phosphorylated bleaching intermediate, phosphorylated opsin, and phosphorylated and regenerated pigment. We also examined the effect of pigment regeneration on the dephosphorylation. The results showed that the dephosphorylation does not show substrate preference in the regeneration cycle and suggested that the dephosphorylation takes place constantly. The results also suggest that, under bright light, some of the regenerated visual pigment remains phosphorylated to reduce the light sensitivity in cones.

Phosphorylation-independent suppression of light-activated visual pigment by arrestin in carp rods and cones.

Visual pigment in photoreceptors is activated by light. Activated visual pigment (R*) is believed to be inactivated by phosphorylation of R* with subsequent binding of arrestin. There are two types of photoreceptors, rods and cones, in the vertebrate retina, and they express different subtypes of arrestin, rod and cone type. To understand the difference in the function between rod- and cone-type arrestin, we first identified the subtype of arrestins expressed in rods and cones in carp retina. We found that two rod-type arrestins, rArr1 and rArr2, are co-expressed in a rod and that a cone-type arrestin, cArr1, is expressed in blue- and UV-sensitive cones; the other cone-type arrestin, cArr2, is expressed in red- and green-sensitive cones. We quantified each arrestin subtype and estimated its concentration in the outer segment of a rod or a cone in the dark; they were ∼0.25 mm (rArr1 plus rArr2) in a rod and 0.6-0.8 mm (cArr1 or cArr2) in a cone. The effect of each arrestin was examined. In contrast to previous studies, both rod and cone arrestins suppressed the activation of transducin in the absence of visual pigment phosphorylation, and all of the arrestins examined (rArr1, rArr2, and cArr2) bound transiently to most probably nonphosphorylated R*. One rod arrestin, rArr2, bound firmly to phosphorylated pigment, and the other two, rArr1 and cArr2, once bound to phosphorylated R* but dissociated from it during incubation. Our results suggested a novel mechanism of arrestin effect on the suppression of the R* activity in both rods and cones.

RDH13L, an enzyme responsible for the aldehyde-alcohol redox coupling reaction (AL-OL coupling reaction) to supply 11-cis retinal in the carp cone retinoid cycle.

Cone photoreceptors require effective pigment regeneration mechanisms to maintain their sensitivity in the light. Our previous studies in carp cones suggested the presence of an unconventional and very effective mechanism to produce 11-cis retinal, the necessary component in pigment regeneration. In this reaction (aldehyde-alcohol redox coupling reaction, AL-OL coupling reaction), formation of 11-cis retinal, i.e. oxidation of 11-cis retinol is coupled to reduction of an aldehyde at a 1:1 molar ratio without exogenous NADP(H) which is usually required in this kind of reaction. Here, we identified carp retinol dehydrogenase 13-like (RDH13L) as an enzyme catalyzing the AL-OL coupling reaction. RDH13L was partially purified from purified carp cones, identified as a candidate protein, and its AL-OL coupling activity was confirmed using recombinant RDH13L. We further examined the substrate specificity, subcellular localization, and expression level of RDH13L. Based on these results, we concluded that RDH13L contributes to a significant part, but not all, of the AL-OL coupling activity in carp cones. RDH13L contained tightly bound NADP(+) which presumably functions as a cofactor in the reaction. Mouse RDH14, a mouse homolog of carp RDH13L, also showed the AL-OL coupling activity. Interestingly, although carp cone membranes, carp RDH13L and mouse RDH14 all showed the coupling activity at 15-37 °C, they also showed a conventional NADP(+)-dependent 11-cis retinol oxidation activity above 25 °C without addition of aldehydes. This dual mechanism of 11-cis retinal synthesis attained by carp RDH13L and mouse RDH14 probably contribute to effective pigment regeneration in cones that function in the light.

Difference in the gain in the phototransduction cascade between rods and cones in carp.

In the vertebrate retina, there are two types of photoreceptors, rods and cones. Rods are highly light-sensitive and cones are less light-sensitive. One of the possible mechanisms accounting for the lower light-sensitivity in cones would be lower signal amplification, i.e., lower gain in the phototransduction cascade in cones. In this study, we compared the difference in the gain between rods and cones electrophysiologically in carp. The initial rising phases of the light responses were analyzed to determine an index of the gain, G, a parameter that can be used to compare the gain among cells of varying outer segment volumes. G (in fL · sec(-2)) was 91.2 ± 14.8 (n = 5) in carp rods and 25.3 ± 3.2 (n = 4) in carp red cones, so that the gain in carp red cones is ∼1/4 of that in carp rods. G was also determined in bullfrog rods and was 81.0 ± 17.2 (n = 3) which was very similar to that in carp rods. The difference in the gain between rods and cones in carp determined in this study (∼1/4 in cones compared with rods) is consistent with that we recently determined biochemically (∼1/5 in cones compared with rods). Together with the result obtained in bullfrog rods in this study and the results obtained by others, we concluded that the gain in the cascade is several-fold lower in cones than in rods in carp and probably in other animal species also.

Quantitative aspects of cGMP phosphodiesterase activation in carp rods and cones.

Cones are less light-sensitive than rods. We showed previously in carp that more light (>100-fold) is required in cones than in rods to activate 50% of cGMP phosphodiesterase (PDE). The lower effectiveness of PDE activation in carp cones is due partly to the fact that the activation rate of transducin (Tr) by light-activated visual pigment (R*) is 5-fold lower in carp cones than in rods. In this study, we tried to explain the remaining difference. First, we examined the efficiency of activation of PDE by activated Tr (Tr*). By activating PDE with known concentrations of the active (guanosine 5'-Ο-(γ-thio)triphosphate (GTPγS)-bound) form of Tr*, we found that Tr* activated PDE at a similar efficiency in rods and cones. Next, we examined the contribution of R* and Tr* lifetimes. In a comparison of PDE activation in the presence (with GTP) and absence (with GTPγS) of Tr* inactivation, PDE activation required more light (and was therefore less effective) when Tr* was inactivated in both rod and cone membranes. This is probably because inactivation of Tr* shortened its lifetime, thereby reducing the number of activated PDE molecules. The effect of Tr* inactivation was larger in cones, probably because the lifetime of Tr* is shorter in cones than in rods. The shorter lifetimes of Tr* and R* in cones seem to explain the remaining difference in the effectiveness of PDE activation between rods and cones.

Substrate specificity and subcellular localization of the aldehyde-alcohol redox-coupling reaction in carp cones.

Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.

Different phosphorylation rates among vertebrate cone visual pigments with different spectral sensitivities.

Cone photoreceptor subtypes having different spectral sensitivities exhibit different recovery kinetics in their photoresponses in some vertebrates. Phosphorylation by G protein-coupled receptor kinase (GRK) is essential for the rapid inactivation of light-activated visual pigment, which is the rate-limiting step of the cone photoresponse recovery in salamander. In this study we compared the rate of light-dependent phosphorylation by GRK7 of carp green- and blue-sensitive cone visual pigments. Blue pigment was phosphorylated significantly less effectively than green pigment, suggesting that the difference in the pigment phosphorylation rate is responsible for the difference in photoresponse kinetics among cone photoreceptor subtypes.

Low activation and fast inactivation of transducin in carp cones.

Cone photoreceptors show lower light sensitivity and briefer light responses than rod photoreceptors. The light detection signal in these cells is amplified through a phototransduction cascade. The first step of amplification in the cascade is the activation of a GTP-binding protein, transducin (Tr), by light-activated visual pigment (R*). We quantified transducin activation by measuring the binding of GTPγS in purified carp rod and cone membrane preparations with the use of a rapid quench apparatus and found that transducin activation by an R* molecule is ∼5 times less efficient in cones than in rods. Transducin activation terminated in less than 1 s in cones, more quickly than in rods. The rate of GTP hydrolysis in Tr*, and thus the rate of Tr* inactivation, was ∼25 times higher in cones than in rods. This faster inactivation of Tr* ensures briefer light responses in cones. The expression level of RGS9 was found to be ∼20 times higher in cones than in rods, which explains higher GTP hydrolytic activity and, thus, faster Tr* inactivation in cones than in rods. Although carp rods and cones express rod- or cone-versions of visual pigment and transducin, these molecules themselves do not seem to induce the differences significantly in the transducin activation and Tr* inactivation in rods and cones. Instead, the differences seem to be brought about in a rod or cone cell-type specific manner.

Multiple mechanisms in the perception of face gender: Effect of sex-irrelevant features.

Effects of sex-relevant and sex-irrelevant facial features on the evaluation of facial gender were investigated. Participants rated masculinity of 48 male facial photographs and femininity of 48 female facial photographs. Eighty feature points were measured on each of the facial photographs. Using a generalized Procrustes analysis, facial shapes were converted into multidimensional vectors, with the average face as a starting point. Each vector was decomposed into a sex-relevant subvector and a sex-irrelevant subvector which were, respectively, parallel and orthogonal to the main male-female axis. Principal components analysis (PCA) was performed on the sex-irrelevant subvectors. One principal component was negatively correlated with both perceived masculinity and femininity, and another was correlated only with femininity, though both components were orthogonal to the male-female dimension (and thus by definition sex-irrelevant). These results indicate that evaluation of facial gender depends on sex-irrelevant as well as sex-relevant facial features.

Larger inhibition of visual pigment kinase in cones than in rods.

In the carp retina, visual pigment kinase, GRK1 (G-protein coupled receptor kinase 1) in rods and GRK7 in cones, is inhibited by a photoreceptor neuronal Ca(2+)-sensor protein, S-modulin (or recoverin) in rods and visinin (formerly named s26) in cones. Here, we compared Ca(2+)-dependent inhibition of GRK1 by S-modulin and that of GRK7 by visinin. First, the concentrations of S-modulin and visinin in the outer segment were estimated: the concentration of visinin (1.2 mM) was 20 times higher than that of S-modulin (53 µM). Based on the determined concentrations of the Ca(2+)-sensor proteins and the known dark Ca(2+) concentrations, we estimated that in situ Ca(2+)-dependent inhibition on GRK in cones would be 2.5 times higher than that in rods at the Ca(2+) concentration in the dark. Because GRK activity is approximately 100 times higher in cones than in rods [Proc. Natl Acad. Sci. USA 102 (2005) 21359], the range of Ca(2+)-dependent inhibition on GRK activity is more than 100 times wider in cones than in rods. The inhibitory effects of S-modulin and visinin on photoreceptor GRKs were indistinguishable, although these Ca(2+)-sensor proteins are expressed in a cell-type specific manner. The inhibition by these Ca(2+)-sensor proteins was slightly higher on GRK7 than GRK1 probably because of a characteristic specific to GRK7.

Dicalcin inhibits fertilization through its binding to a glycoprotein in the egg envelope in Xenopus laevis.

Fertilization comprises oligosaccharide-mediated sperm-egg interactions, including sperm binding to an extracellular egg envelope, sperm penetration through the envelope, and fusion with an egg plasma membrane. We show that Xenopus dicalcin, an S100-like Ca(2+)-binding protein, present in the extracellular egg envelope (vitelline envelope (VE)), is a suppressive mediator of sperm-egg interaction. Preincubation with specific antibody greatly increased the efficiency of in vitro fertilization, whereas prior application of exogenous dicalcin substantially inhibited fertilization as well as sperm binding to an egg and in vitro sperm penetration through the VE protein layer. Dicalcin showed binding to protein cores of gp41 and gp37, constituents of VE, in a Ca(2+)-dependent manner and increased in vivo reactivity of VE with a lectin, Ricinus communis agglutinin I, which was accounted for by increased binding ability of gp41 to the lectin and greater exposure of gp41 to an external environment. Our findings strongly suggest that dicalcin regulates the distribution of oligosaccharides within the VE through its binding to the protein core of gp41, probably by modulating configuration of oligosaccharides on gp41 and the three-dimensional structure of VE framework, and thereby plays a pivotal role in sperm-egg interactions during fertilization.

High cGMP synthetic activity in carp cones.

Cones show briefer light responses than rods and do not saturate even under very bright light. Using purified rod and cone homogenates, we measured the activity of guanylate cyclase (GC), an enzyme responsible for cGMP synthesis and therefore recovery of a light response. The basal GC activity was 36 times higher in cones than in rods: It was mainly caused by higher expression levels of GC in cones (GC-C) than in rods (GC-R). With identification and quantification of GC-activating protein (GCAP) subtypes expressed in rods and cones together with determination of kinetic parameters of GC activation in the presence and absence of GCAP, we estimated the in situ GC activity in rods and cones at low and high Ca(2+) concentrations. It was revealed that the GC activity would be >10 times higher in cones than in rods in both the dark-adapted and the light-adapted states. Electrophysiological estimation of the GC activity measured in the truncated preparations of rod and cone outer segments gave consistent results. Our estimation of the in situ GC activity reasonably explained the rapid recovery and nonsaturating behavior of cone light responses.

Averageness or symmetry: which is more important for facial attractiveness?

Effects of averageness and symmetry on the judgment of facial attractiveness were investigated using a generalized Procrustes method and multiple regression analyses. Participants (n=114) rated attractiveness of 96 photographs of faces with neutral expressions. Through a generalized Procrustes method, the faces and their mirror-reversed versions were represented as points on a hyperplane. Both averageness and symmetry of each individual were defined as distances on the plane. A multiple regression analysis was performed to examine the effect of symmetry and averageness for each gender. For male faces, both symmetry and averageness affected attractiveness ratings positively , and there was no difference between the effects of averageness and symmetry. On the other hand, for female faces only averageness affected attractiveness, whereas symmetry did not. However, these effects were not large.